Anti-HTRA-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for HTRA-2 on a Western blot?

HTRA-2 migrates as a ~49 kDa band corresponding to the mature processed form found in mitochondria. The full-length precursor is 458 amino acids (~52 kDa) but undergoes N-terminal cleavage of the mitochondrial targeting sequence upon import into the intermembrane space. Depending on your sample preparation and cell apoptotic state, you may observe both the precursor and mature forms. Under reducing conditions with standard SDS-PAGE, the mature processed form at 49 kDa is most commonly detected in mitochondrial or whole-cell lysates.

What starting dilution do you recommend for HTRA-2 Western blot detection?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody. This dilution typically provides robust signal from mitochondrial-enriched fractions or whole-cell lysates prepared from apoptotic cells where HTRA-2 is released. If working with cytosolic fractions from apoptotic cells, you may achieve adequate signal at 1:2000. For less abundant samples or non-apoptotic whole-cell lysates, begin at 1:500 and titrate upward. Overnight incubation at 4°C often improves sensitivity compared to one-hour room-temperature protocols.

Is this HTRA-2 antibody validated in rat or mouse samples?

This antibody is validated in human samples. Rat cross-reactivity is predicted based on high sequence homology in the immunogenic region, but we have not formally validated performance in rat lysates. Mouse HTRA-2 shares approximately 85 percent identity with human in key epitope regions; however, we cannot guarantee recognition without empirical testing. If working with rodent models, we recommend running a positive control human lysate alongside your samples and verifying band migration at the expected molecular weight. Contact us if you obtain rodent validation data.

Does HTRA-2 require special sample preparation or enrichment for Western blot?

HTRA-2 is a mitochondrial intermembrane space protein, so mitochondrial enrichment improves sensitivity, though whole-cell lysates often suffice. Standard RIPA or NP-40 lysis buffers work well; avoid overly harsh detergents that may denature the epitope. If studying apoptosis, HTRA-2 is released into the cytosol, making cytosolic fractions ideal. Include protease inhibitors during lysis to prevent auto-proteolysis, as HTRA-2 is an active serine protease. For non-apoptotic cells, consider inducing mild stress or using mitochondrial isolation kits to concentrate signal.

Can this antibody detect HTRA-2 in immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are broadly validated for immunofluorescence and immunohistochemistry in addition to Western blot. For IF, HTRA-2 localizes to mitochondria in healthy cells, appearing as punctate perinuclear and cytoplasmic staining that co-localizes with mitochondrial markers like Tom20 or cytochrome c. During apoptosis, staining becomes diffuse as HTRA-2 is released. Start at 1:100 to 1:200 dilution and optimize on your cell type. For IHC on paraffin sections, antigen retrieval with citrate buffer is generally recommended. Titrate the antibody empirically for your fixation protocol.

What positive control lysate should I use for HTRA-2 Western blot experiments?

HeLa or HEK293T whole-cell lysates serve as reliable positive controls for HTRA-2 detection. To enrich signal, treat cells with apoptosis inducers such as staurosporine (1 µM, 4-6 hours) or etoposide before lysis; HTRA-2 is released from mitochondria during apoptosis and becomes more abundant in whole-cell preparations. Mitochondrial fractions from untreated cells also work well. Load 20-30 µg total protein per lane. Negative controls include HTRA-2 knockout cell lines if available, or pre-absorption with recombinant HTRA-2 protein to confirm specificity.

How should I store this HTRA-2 antibody and what is the expected shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is sufficient for multiple experiments when aliquoted upon arrival. Polyclonal antibodies from Triple Point Biologics typically retain activity for at least two years when stored properly. For working stocks used within two weeks, 4°C storage in the presence of 0.02 percent sodium azide is acceptable. Avoid prolonged exposure to room temperature and do not store diluted antibody for more than one week.

Does HTRA-2 have isoforms or splice variants I should be aware of?

HTRA-2 is primarily expressed as a single major isoform, the 458-amino acid precursor encoded by the HTRA2 gene on chromosome 2. Alternative splicing has not been widely reported in the literature, and most studies focus on the canonical mitochondrial form. Post-translational processing removes the N-terminal mitochondrial targeting sequence, yielding the mature active protease found in the intermembrane space. Some reports describe S-nitrosylation or phosphorylation under stress conditions, which may subtly alter migration, but these modifications are not routinely resolved on standard SDS-PAGE. Expect one predominant band in most experimental systems.